Análise da microbiota intestinal em adultos com hábitos alimentares distintos e de associações com a inflamação e resistência à insulina

Introdução: A microbiota intestinal possui grande diversidade de bactérias, predominantemente dos filos Bacteroidetes e Firmicutes, com múltiplas funções. A alimentação pode alterar sua composição e função. Alto teor de gordura saturada altera a permeabilidade intestinal, eleva os lipopolissacarídeos e predispõe à inflamação subclínica crônica. Dieta rica em fibras, como a vegetariana, induz elevação de ácidos graxos de cadeia curta e benefícios metabólicos. Objetivos: Para analisar a composição da microbiota intestinal de adventistas com diferentes hábitos alimentares e associá-los à inflamação subclínica e resistência à insulina, esta tese incluiu: 1) revisão dos mecanismos que associam a alimentação à microbiota intestinal e ao risco cardiometabólico; 2) verificação da composição da microbiota intestinal segundo diferentes hábitos alimentares e de associações com biomarcadores de doenças cardiometabólicas; 3) avaliação da associação entre a abundância de Akkermansia muciniphila e o metabolismo da glicose; 4) análise da presença de enterótipos e de associações com características clínicas. Métodos: Este estudo transversal incluiu 295 adventistas estratificados segundo hábitos alimentares (vegetariano estrito, ovo-lacto-vegetariano e onívoro). Foram avaliadas associações com dados clínicos, bioquímicos e inflamatórios. O perfil da microbiota foi obtido por sequenciamento do gene 16S rRNA (Illumina® Miseq). Resultados: 1) Há evidências de que as relações entre dieta, inflamação, resistência à insulina e risco cardiometabólico são em parte mediadas pela composição da microbiota intestinal. 2) Vegetarianos apresentaram melhor perfil clínico quando comparados aos onívoros. Confirmou-se maior abundância de Firmicutes e Bacteroidetes, que não diferiram segundo a adiposidade corporal. Entretanto, vegetarianos estritos apresentaram mais Bacteroidetes, menos Firmicutes e maior abundância do gênero Prevotella quando comparados aos outros dois grupos de hábitos alimentares. Entre os ovo-lactovegetarianos verificou-se maior proporção de Firmicutes especialmente do gênero Faecalibacterium. Nos onívoros, houve super-representação do filo Proteobacteria (Succinivibrio e Halomonas) comparados aos vegetarianos. 3) Indivíduos normoglicêmicos apresentaram maior abundância de Akkermansia muciniphila que aqueles com glicemia alterada. A abundância desta bactéria correlacionou-se inversamente à glicemia e hemoglobina glicosilada. 4) Foram identificados três enterótipos (Bacteroides, Prevotella e Ruminococcaceae), similares àqueles previamente descritos. As concentrações de LDL-C foram menores no enterótipo 2, no qual houve maior frequência de vegetarianos estritos. Discussão: 1) Conhecimentos sobre participação da microbiota na fisiopatologia de doenças poderão reverter em estratégias para manipulá-la para promover saúde. 2) Apoia-se a hipótese de que hábitos alimentares se associam favorável ou desfavoravelmente a características metabólicas e inflamatórias do hospedeiro via alterações na composição da microbiota intestinal. Sugerimos que a exposição a alimentos de origem animal possa impactar negativamente nas proporções de comunidades bacterianas. 3) Sugerimos que a abundância da Akkermansia muciniphila possa participar do metabolismo da glicose. 4) Reforçamos que a existência de três enterótipos não deva ser específica de certas populações/continentes. Apesar de desconhecido o significado biológico destes agrupamentos, as correlações com o perfil lipídico podem sugerir sua utilidade na avaliação do risco cardiometabólico. Conclusões: Nossos achados fortalecem a ideia de que a composição da microbiota intestinal se altera mediante diferentes hábitos alimentares, que, por sua vez, estão associados a alterações nos perfis metabólicos e inflamatórios. Estudos prospectivos deverão investigar o potencial da dieta na prevenção de distúrbios cardiometabólicos mediados pela microbiota.

Single reaction interface flow system for chemiluminescent monitoring of mannitol based on its hydroxyl radical scavenger activity

A single reaction interface flow analysis (SIFA) system for the monitoring of mannitol in pharmaceutical formulations and human urine is presented. The developed approach takes advantage of the mannitol scavenger aptitude to inhibit the chemiluminescent reaction between luminol and myoglobin in the absence of H(2)O(2). The SIFA system facilitated the fully automation of the developed methodology, allowing the in-line reproducible handling of chemical species with a very short lifetime as is the case of the hydroxyl radical generated in the abovementioned luminol/myoglobin reaction. The proposed methodology allowed the determination of mannitol concentrations between 25 mmol L(-1) and 1 mol L(-1), with good precision (R.S.D. < 4.7%, n = 3) and a sampling frequency of about 60 h(-1). The procedure was applied to the determination of mannitol in pharmaceuticals and in human urine samples Without any pretreatment process. The results obtained for pharmaceutical formulations were statistically comparable to those provided by the reference method (R.D. < 4.6%); recoveries values obtained in the analysis of spiked urine samples (between 94.9 and 105.3% of the added amount) were also satisfactory. (C) 2008 Elsevier B.V. All rights reserved.

Protective effect of octreotide and infliximab in an experimental model of indomethacin-induced inflammatory bowel disease

Indomethacin administration in animals increases permeability of the small intestine, leading to inflammation that mimics Crohn`s disease. Nonsteroidal anti-inflammatory drugs increase the permeability of the intestinal epithelial barrier and should therefore be used with caution in patients with Crohn`s disease. We analyzed the protective effects of octreotide and the tumor necrosis factor-alpha inhibitor infliximab in a rat model of indomethacin-induced enterocolitis. Male Wistar rats received 20 mg of infliximab or 10 mu g of octreotide 24 h prior to injection with indomethacin. Intestinal permeability was analyzed using Cr-51-ethylenediaminetetraacetic acid clearance. No microscopic or macroscopic alterations were observed in the rats receiving infliximab or octreotide, both of which increased permeability (P < 0.001 versus controls). Our macroscopic and microscopic findings might be related to the low specificity of infliximab and suggest that cytokines affect the intestinal epithelial barrier, as evidenced by the protective effect that infliximab had on the permeability parameters evaluated.

Early antibiotic administration affects the gut barrier function and the immune response to oral antigen in suckling rats

Abstract: The increasingly high hygienic standards characterizing westernized societies correlate with an increasingly high prevalence of allergic disease. Initially based on these observations, the hygiene hypothesis postulates that reduced microbial stimulation during infancy impairs the immune system development and increases the risk of allergy. Moreover, there is increasing evidence that the crosstalk existing between the intestine and the resident microbiota is crucial for gut homeostasis. In particular, bacterial colonization of the gut affects the integrity of the gut barrier and stimulates the development of the gut associated immune tissue, both phenomena being essential for the immune system to mount a controlled response to food antigens. Therefore, alterations in the microbial colonization process, by compromising the barrier homeostasis, may increase the risk of food allergy. In this context, antibiotic treatment, frequently prescribed during infancy, affects gut colonization by bacteria. However, little is known about the impact of alterations in the colonization process on the maturation of the gut barrier and on the immunological response to oral antigens. The objective of this work was to determine the impact of a commercial antibiotic preparation employed in pediatric settings on the gut barrier status at the critical period of the suckling/weaning transition and to evaluate the physiological consequences of this treatment in terms of immune response to food antigens. We established an antibiotic-treated suckling rat model relevant to the pediatric population in terms of type, dose and route of administration of the antibiotic and of changes in the patterns of microbial colonization. Oral tolerance to a novel luminal antigen (ovalbumin) was impaired when the antigen was introduced during antibiotic treatment. These results paralleled to alterations in the intestinal permeability to macromolecules and reduced intestinal expression of genes coding for the major histocomptatibility complex II molecules, which suggest a reduced capacity of antigen handling and presentation in the intestine of the antibiotic-treated animals. In addition, low luminal IgA levels and reduced intestinal expression of genes coding for antimicrobial proteins suggest that protection against pathogens was reduced under antibiotic treatment. In conclusion, we observed in suckling rats that treatment with abroad-spectrum antibiotic commonly used in pediatric practices reduced the capacity of the immune system to develop tolerance. The impact of the antibiotic treatment on the immune response to the antigen-was likely mediated by the alterations of the gut microbiota, through impairment in the mechanisms of antigen handling and presentation. This work reinforces the body of data supporting a key role of the intestinal microbiota modulating the risk of allergy development and leads us to propose that the introduction of new food antigens should be avoided during antibiotic treatment in infants. Résumé: L'augmentation du niveau d'hygiène caractérisant les sociétés occidentales semble être fortement corrélée avec l'augmentation des cas d'allergie dans ces pays. De cette observation est née l'hypothèse qu'une diminution des stimuli microbiens pendant l'enfance modifie le développement du système immunitaire augmentant ainsi le risque d'allergie. En ce sens, un nombre croissant de données indiquent que les interactions existant entre l'intestin et les bactéries résidantes sont cruciales pour l'équilibre du système. En effet, la présence de bactéries dans l'intestin affecte l'intégrité de sa fonction de barrière et stimule le développement du système immunitaire intestinal. Ces deux paramètres étant essentiels à la mise en place d'une réponse contrôlée vis à vis d'un antigène reçu oralement, toute modification du processus naturel de colonisation compromettant l'équilibre intestinal pourrait augmenter le risque d'allergie. Les traitements aux antibiotiques, fréquemment prescrits en pédiatrie, modifient de façon conséquente le processus de colonisation bactérienne. Cependant peu de données existent concernant l'impact d'une altération du processus de colonisation sur la maturation de la barrière intestinale et de la réponse immunitaire dirigée contre un antigène. L'objectif de ce travail était de déterminer l'impact d'un antibiotique commercial et employé en pédiatrie sur l'état de la barrière intestinale au moment critique du sevrage et d'évaluer les conséquences physiologiques d'un tel traitement sur la réponse immune à un antigène alimentaire. Nous avons mis en place un modèle de rats allaités, traités à l'antibiotique, le plus proche possible des pratiques pédiatriques, en terme de nature, dose et voie d'administration de l'antibiotique. Nous avons constaté que l'établissement de la tolérance orale à un nouvel antigène (l'ovalbumine) est altéré quand celui-ci est donné pour la première fois au cours du traitement antibiotique. Ces résultats coïncident avec une diminution de la perméabilité intestinale aux macromolécules, ainsi qu'avec une diminution de l'expression des gènes codant pour les molécules du complexe majeur d'histocomptatibilité de classe II, suggérant une modification de l'apprêtement et de la présentation de l'antigène au niveau intestinal chez les rats traités à l'antibiotique. De plus, un faible taux d'IgA et une diminution de l'expression des gènes codant pour des protéines antimicrobiennes, observés après l'administration d'antibiotique, laissent à penser que la protection contre un pathogène est diminuée lors d'un traitement antibiotique. En conclusion, nous avons observé qu'un traitement antibiotique à large spectre d'activité, couramment utilisé en pédiatrie, réduit la capacité d'induction de la tolérance orale chez le rat allaité. L'impact du traitement antibiotique sur la réponse immune semble induite par l'altération de la flore intestinale via son effet sur les mécanismes d'apprêtement et de présentation de l'antigène. Ce travail renforce l'ensemble des données existantes qui accorde à la flore intestinale un rôle clef dans la modulation du risque de développement d'allergie et nous amène à recommander d'éviter l'introduction d'un nouvel aliment lorsqu'un enfant est traité aux antibiotiques.

Systemic antibody responses to gut commensal bacteria during chronic HIV-1 infection.

BACKGROUND: Human systemic antibody responses to commensal microbiota are not well characterised during health and disease. Of particular interest is the analysis of their potential modulation caused by chronic HIV-1 infection which is associated with sustained enteropathy and systemic B cell disturbances reflected by impaired B cell responses and chronic B cell hyperactivity. The mechanisms underlying B cell hyperactivation and the specificities of the resulting hypergammaglobulinaemia are only poorly understood. METHODS: By a technique referred to as live bacterial FACS (fluorescence-activated cell sorting), the present study investigated systemic antibody responses to several gut and skin commensal bacteria as well as Candida albicans in longitudinal plasma and serum samples from healthy donors, chronic HIV-1-infected individuals with or without diarrhoea and patients with inflammatory bowel disease (IBD). RESULTS: The data show that systemic antibody responses to the commensal microbiota were abundantly present in humans and remained remarkably stable over years. Overall systemic antibody responses to gut commensal bacteria were not affected during chronic HIV-1 infection, with titres decreasing when normalised to elevated plasma immunoglobulin G (IgG) levels found in patients with HIV. In contrast, increases in the titres of high affinity antimicrobiota antibodies were detected in patients with IBD, demonstrating that conditions with known increased intestinal permeability and aberrant mutualism can induce changes in antibody titres observed in these assays. CONCLUSION: Neither HIV-associated enteropathy nor B cell dysfunction impact on the high-affinity systemic antibody responses to gut commensal bacteria. HIV-associated hypergammaglobulinaemia is therefore unlikely to be driven by induction of antimicrobiota antibodies.

Toll-Interacting Protein Modulates Gut Flora Composition, Epithelial Barrier Integrity and Susceptibility to Colitis

Toll-like receptor ( TLR) s ignals are key to maintaining hostmicrobial i nteractions. T he T oll-interacting-protein (Tollip) is a ubiquitously-expressed inhibitor of inflammasome a nd TLR signaling. W e hypothesized that T ollip might control g ut homeostasis. G enetic ablation of T ollip d id not lead to spontaneous colitis b ut h ad d ramatic c onsequences on t he intestinal expression of the α-defensin cryptidin 4 and the C-type lectin R EGIIIβ. These c hanges were associated with intestinal dysbiosis a nd e nhanced colonization b y segmented filamentous bacteria - a k ey p ro-inflammatory component of the microbiota. Tollip deficiency increased susceptibility to dextran sulfate sodium (DSS) colitis and aggravated chronic Th17-driven colitis in IL-10-/- mice. Flora d epletion w ith a ntibiotics in T ollip-/- mice w as not sufficient to restore DSS colitis susceptibility and deletion of Tollip in n on-hematopoietic c ells using bone-marrow chimeras w as sufficient to increase s usceptibility t o DSS colitis. After D SS administration, we o bserved several e pithelial defects i n Tollip-/- mice including early tight junctions disruption, increased epithelial apoptosis, and increased intestinal permeability. Overall, our data show that T ollip significantly impacts intestinal h omeostasis by controlling b acterial ecology and intestinal r esponse to chemical and immunological stresses.

Toll-interacting protein modulates colitis susceptibility in mice.

BACKGROUND: The intestinal epithelium accommodates with a myriad of commensals to maintain immunological homeostasis, but the underlying mechanisms regulating epithelial responsiveness to flora-derived signals remain poorly understood. Herein, we sought to determine the role of the Toll/interleukin (IL)-1 receptor regulator Toll-interacting protein (Tollip) in intestinal homeostasis. METHODS: Colitis susceptibility was determined after oral dextran sulfate sodium (DSS) administration or by breeding Tollip on an IL-10 background. The intestinal flora was depleted with 4 antibiotics before DSS exposure to assess its contribution in colitis onset. Bone marrow chimeras were generated to identify the cellular compartment, whereby Tollip may negatively regulate intestinal inflammation in response to DSS. Tollip-dependent epithelial barrier functions were studied in vitro by using Tollip-knockdown in Caco-2 cells and in vivo by immunohistochemistry and fluorescein isothiocyanate-labeled dextran gavage. RESULTS: Genetic ablation of Tollip did not lead to spontaneous intestinal inflammatory disorders. However, Tollip deficiency aggravated spontaneous disease onset in IL-10 mice and increased susceptibility to DSS colitis. Increased colitis severity in Tollip-deficient mice was not improved by bacterial flora depletion using broad-spectrum antibiotics. In addition, DSS exposure of bone marrow chimeric mice revealed a protective role for Tollip in nonhematopoietic cells. Knockdown of Tollip in epithelial cells led to exaggerated NFκ-B activity and proinflammatory cytokine secretion. Finally, DSS-treated Tollip mice showed enhanced intestinal permeability and increased epithelial apoptosis when compared with wild-type controls, a finding that coincided with tight junction alterations on injury. CONCLUSION: Overall, our data show an essential role for Tollip on colitis susceptibility in mice.

Effect of a selective nonsteroidal anti-inflammatory inhibitor of cyclooxygenase-2 on the small bowel of rats

The pathogenesis of nonsteroidal anti-inflammatory drug (NSAID) enteropathy is a complex process involving the uncoupling of mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase (COX). Rofecoxib, a selective inhibitor of COX-2, has shown less gastric damage, but the same beneficial effect is not clear in the case of the small bowel. Fifty-seven male Wistar rats (250-350 g) were divided into three groups (N = 19 each) to evaluate the effect of this NSAID on the rat intestine. The groups received 2.5 mg/kg rofecoxib, 7.5 mg/kg indomethacin or water with 5% DMSO (control) given as a single dose by gavage 24 h before the beginning of the experiment. A macroscopic score was used to quantify intestinal lesions and intestinal permeability was measured using [51Cr]-ethylenediaminetetraacetic acid ([51Cr]-EDTA). The extent of intestinal lesion, indicated by a macroscopic score, was significantly lower when rofecoxib was administered compared to indomethacin (rofecoxib = 0.0 vs indomethacin = 63.6 ± 25.9; P < 0.05) and did not differ from control. The intestinal permeability to [51Cr]-EDTA was significantly increased after indomethacin (control = 1.82 ± 0.4 vs indomethacin = 9.12 ± 0.8%; P < 0.0001), but not after rofecoxib, whose effect did not differ significantly from control (control = 1.82 ± 0.4 vs rofecoxib = 2.17 ± 0.4%; ns), but was significantly different from indomethacin (indomethacin = 9.12 ± 0.8 vs rofecoxib = 2.17 ± 0.4%; P < 0.001). In conclusion, the present data show that rofecoxib is safer than indomethacin in rats because it does not induce macroscopic intestinal damage or increased intestinal permeability.

Correlation between symptoms of the irritable bowel syndrome and the response to the food extract skin prick test

The relationship between the irritable bowel syndrome (IBS) and food intolerance is not clear. We studied the cutaneous response to food antigens in 43 volunteers who were students and employees of the Faculty of Medicine of Universidade Federal Fluminense. Subjects were divided into 3 groups after evaluation for Roma II criteria for functional disease of the gastrointestinal tract: group I, 14 volunteers with IBS; group II, 15 volunteers with functional dyspepsia; group III, 14 volunteers without habitual gastrointestinal symptoms. The subjects were submitted to the skin prick test with 9 food antigen extracts, for a total of 387 skin tests (9 per volunteer). Of the 126 tests applied to group I, 24 (19.4%) were positive (a 3-mm wider papule than the negative control) and of the 135 tests applied to group II, 3 (2.3%) were positive. Of the 126 tests applied to group III, 6 (4%) were positive. The number of positive responses obtained in group I (IBS) differed significantly from the other 2 groups (P < 0.01). None of the volunteers with IBS reported intolerance to any isolated food. The higher reactivity to food antigens in group I compared to groups II and III suggests that intestinal permeability may be increased in patients with IBS.

Optimisation et évaluation d’un nouveau test PAMPA amélioré pour la prédiction de l’absorption intestinale de médicaments.

Ce mémoire rapporte l’optimisation et l’évaluation d’une nouvelle version du test PAMPA (Parallel Artificial Membrane Permeability Assay) appelée Néo-PAMPA. Ce test qui permet la prédiction de l’absorption intestinale de médicaments consiste en l’utilisation d’une membrane modèle de la paroi intestinale composée d’une bicouche lipidique déposée sur un coussin de polydopamine recouvrant un filtre poreux. En effet, nous nous sommes intéressés lors de ce projet à la mise en place d’une membrane artificielle qui serait plus représentative de la paroi intestinale humaine. Nous avons pu déterminer, suite à une étude comparative des propriétés de huit médicaments ainsi que les coefficients de perméabilité obtenus, que les filtres en polycarbonate présentaient le meilleur choix de support solide pour la membrane. Nous avons également vérifié la déposition du coussin de polydopamine qui apporte le caractère fluide à la bicouche lipidique. Les résultats des tests de perméabilité ont démontré que le coussin de polymère n’obstrue pas les pores du filtre après un dépôt de 4h. Nous avons par la suite étudié la déposition de la bicouche lipidique sur le filtre recouvert de polydopamine. Pour ce faire, deux méthodes de préparation de liposomes ainsi que plusieurs tailles de liposomes ont été testées. Aussi, la composition en phospholipides a été sujette à plusieurs changements. Tous ces travaux d’optimisation ont permis d’aboutir à des liposomes préparés selon la méthode du « film lipidique » à partir d’un mélange de dioléoylphosphatidylcholine (DOPC) et de cholestérol. Une dernière étape d’optimisation de la déposition de la bicouche reste à améliorer. Enfin, le test standard Caco-2, qui consiste à évaluer la perméabilité des médicaments à travers une monocouche de cellules cancéreuses du colon humain, a été implémenté avec succès dans le but de comparer des données de perméabilité avec un test de référence.

L’entérotoxine STb d’Escherichia coli déloge la claudine-1 des jonctions serrées

Escherichia coli produit diverses entérotoxines thermolabiles et thermostables. STb est une toxine de faible poids moléculaire résistant à la chaleur chargée de la diarrhée chez les animaux de la ferme. Une étude antérieure a montré que les cellules ayant internalisé la toxine STb provoquent un dysfonctionnement de la barrière épithéliale par des changements dans les protéines des jonctions serrées (TJ). Ces modifications contribuent probablement à la diarrhée observée. Pour mieux comprendre le mécanisme de l'augmentation de la perméabilité intestinale, nous avons traité les cellules du côlon humain (T84) avec la toxine purifiée STb une fois que les cellules ont été récoltées et les protéines extraites. Après l'utilisation d'une solution contenant 1% de Nonidet P-40 (un détergent non dénaturant, non ionique), nous avons étudié la distribution de la claudine -1, une protéine majeure des TJs, responsable de l'imperméabilité de l'épithélium, entre la membrane (NP40-insoluble) et le cytoplasme (NP40-soluble). En utilisant l’immunoblot et la microscopie confocale, nous avons observé que le traitement des monocouches de cellules T84 avec STb induit la redistribution de la claudine-1. Après 24h, les cellules cultivées en milieu faible en Ca+ (5 uM) et traitées par STb, ont montré qu’environ 40 % de plus de la claudine-1 se sont délogées dans le cytoplasme par comparaison au contrôle. En passant d’un milieu faible à un milieu contenant des quantités physiologiques de Ca++ (1,8 mM) nous avons observé une augmentation du taux de claudine- 1 délogé, comme la délocalisation comparable et ce, après 6h. Un milieu supplémenté avec la même concentration de Mg++ ou Zn++ n'a pas affecté le taux de délogement comparé au milieu contenant une faible teneur en Ca++. En utilisant des anticorps anti-phosphosérine et anti-phosphothréonine, nous avons observé que la perte des claudines-1 de la membrane a été accompagnée par une déphosphorylation de cette protéine des TJs. Dans l'ensemble, nos résultats ont montré une importante redistribution de la claudine-1 dans les cellules traitées par la toxine STb. La perte de la claudine-1 phosphorylée de la membrane est susceptible d'être impliquée dans la perméabilité accrue observée. Les mécanismes par lesquels ces changements sont provoqués restent à élucider.

Validation et conditionnement d'un test PAMPA amélioré pour l'évaluation de la perméabilité membranaire de médicaments.

Les tests PAMPA et les tests Caco-2 sont des essais in vitro de l’évaluation de la perméabilité intestinale des médicaments. Ils sont réalisés lors de la phase de découverte du médicament. Les tests PAMPA ne sont pas biologiquement représentatifs de la paroi intestinale, mais ils sont rapides et peu coûteux. Les tests Caco-2 nécessitent plus de 21 jours pour la culture cellulaire et des installations spécifiques sont requises. Ils sont constitués d’une monocouche d’entérocytes à confluence et donc plus biologiquement représentatifs. Il y a un besoin pour le développement d’un essai qui est biologiquement représentatif de la membrane intestinale humaine, rapide et peu coûteux. Le premier but de ce projet était de développer une méthode analytique qui permettrait l’évaluation simultanée de huit médicaments témoins utilisés pour la validation de l’essai de perméabilité. Le deuxième but de ce projet était donc d’améliorer la membrane des tests PAMPA pour proposer un nouveau test : le néoPAMPA. Contrairement au test PAMPA traditionnel, cette membrane est constituée de trois composantes : (1) un filtre poreux qui agit à titre de support, (2) un coussin polydopamine chargé négativement qui sert d’ancrage et qui assure la fluidité de la bicouche et (3) une bicouche lipidique formée par fusion de vésicules. Une méthode analytique HPLC-MS/MS a été validée selon les spécifications de la FDA et de la EMA. Cette méthode a permis de quantifier simultanément les huit médicaments standards utilisés pour le test néoPAMPA. Le test PAMPA traditionnel a été mis en place à titre d’essai control. Les coefficients de perméabilité mesurés pour les huit médicaments au travers de la membrane PAMPA comparaient favorablement aux résultats de la littérature. Les composantes de la membrane néoPAMPA ont été optimisées. Les conditions optimales retenues étaient les filtres de polycarbonate hydrophile ayant des pores de 15 nm, les plaques Costar 12 puits comme dispositif des tests de perméabilité, une bicouche lipidique composée de 70 % DOPC et de 30 % cholestérol cationique ainsi qu’une déposition des liposomes en présence de 150 mM NaCl suivi d’un équilibre d’1 h en présence d’une solution saturée en DOPC. Les stabilités de la cassette de médicaments et des liposomes sont insuffisantes pour le conditionnement commercial des membranes néoPAMPA. Les différentes optimisations réalisées ont permis d’améliorer la membrane néoPAMPA sans toutefois la rendre fonctionnelle. La membrane néoPAMPA n’est toujours pas en mesure de discriminer des molécules en fonction de leur perméabilité attendue.

The effect of nicotine in vitro on the integrity of tight junctions in Caco-2 cell monolayers

Ulcerative colitis is characterised by impairment of the epithelial barrier and tight junction alterations resulting in increased intestinal permeability. UC is less common in smokers with smoking reported to decrease paracellular permeability. The aim of this study was thus to determine the effect of nicotine, the major constituent in cigarettes and its metabolites on the integrity of tight junctions in Caco-2 cell monolayers. The integrity of Caco-2 tight junctions was analysed by measuring the transepithelial electrical resistance (TER) and by tracing the flux of the fluorescent marker fluorescein, after treatment with various concentrations of nicotine or nicotine metabolites over 48 h. TER was significantly higher compared to the control for all concentrations of nicotine 0.01-10 M at 48 h (p < 0.001), and for 0.01 mu M (p < 0.001) and 0.1 mu M and 10 M nicotine (p < 0.01) at 12 and 24 h. The fluorescein flux results supported those of the TER assay. TER readings for all nicotine metabolites tested were also higher at 24 and 48 h only (p <= 0.01). Western blot analysis demonstrated that nicotine up-regulated the expression of the tight junction proteins occludin and claudin-l (p < 0.01). Overall, it appears that nicotine and its metabolites, at concentrations corresponding to those reported in the blood of smokers, can significantly improve tight junction integrity, and thus, decrease epithelial gut permeability. We have shown that in vitro, nicotine appears more potent than its metabolites in decreasing epithelial gut permeability. We speculate that this enhanced gut barrier may be the result of increased expression of claudin-l and occludin proteins, which are associated with the formation of tight junctions. These findings may help explain the mechanism of action of nicotine treatment and indeed smoking in reducing epithelial gut permeability. (c) 2007 Elsevier Ltd. All rights reserved.

A double-blind, randomized, controlled crossover trial of glutamine supplementation in home parenteral nutrition

Objective: Studies suggest clinical benefit of glutamine-supplemented parenteral nutrition. The aim was to determine if the inclusion of 10 g of glutamine as part of the nitrogen source of home parenteral nutrition (HPN) reduces infectious complications. Subjects/Methods: Thirty-five patients on HPN were recruited and 22 completed the study. Patients were randomized to receive either standard HPN or glutamine-supplemented HPN. Patients were assessed at randomization, 3 and 6 months later then they were crossed over to the alternative HPN and reassessed at 3 and 6 months. Assessments included plasma amino acid concentrations, intestinal permeability and absorption, nutritional status, oral and parenteral intake, quality of life, routine biochemistry and haematology. Results: No difference was seen between the groups at randomization. No difference was detected between the treatment phases for infective complications (55% in the standard treatment phase and 36% in the glutamine-supplemented phase P 0.67). There were no differences in nutritional status, intestinal permeability, plasma glutamine concentrations or quality of life. Conclusion: Although limited by the sample size, the study has shown that glutamine as part of the nitrogen source of parenteral nutrition can be given to patients on HPN for 6 months without any adverse effects.

Dietary supplementation with Bifidobacterium lactis NCC2818 from weaning reduces local immunoglobulin production in lymphoid-associated tissues but increases systemic antibodies in healthy neonates

Weaning is associated with a major shift in the microbial community of the intestine, and this instability may make it more acquiescent than the adult microbiota to long-term changes. Modulation achieved through dietary interventions may have potentially beneficial effects on the developing immune system, which is driven primarily by the microbiota. The specific aim of the present study was to determine whether immune development could be modified by dietary supplementation with the human probiotic Bifidobacterium lactis NCC2818 in a tractable model of weaning in infants. Piglets were reared by their mothers before being weaned onto a solid diet supplemented with B. lactis NCC2818, while sibling controls did not receive supplementation. Probiotic supplementation resulted in a reduction in IgA (P,0·0005) and IgM (P,0·009) production by mucosal tissues but had no effect on IgG production (P.0·05). Probiotic- supplemented pigs had more mast cells than unsupplemented littermates (P,0·0001), although numbers in both groups were low. In addition, the supplemented piglets made stronger serum IgG responses to fed and injected antigens (P,0·05). The present findings are consistent with B. lactis NCC2818 reducing intestinal permeability induced by weaning, and suggest that the piglet is a valuable intermediate between rodent models and human infants. The results also strongly suggest that measures of the effect of probiotic supplementation on the immune system need to be interpreted carefully as proxy measures of health benefit. However, they are useful in developing an understanding of the mechanism of action of probiotic strains, an important factor in predicting favourable health outcomes of nutritional intervention.